Cystatin C was discovered and characterized in 1981 by A. Grubb and H. Lovberg [Grubb A O, et al: Proc. Nati. Acad. Sci. USA 1982;79]. Cystatin C was the first protein discovered in what later has been named the Cystatin protein superfamily. The Cystatin protein superfamily is a group of proteins that inhibits of cystein protease enzymes. Other biological functions of Cystatin C are presently under investigation. Contrary to other members of the Cystatin superfamily, Cystatin C is present in all body fluids, while the total composition of different Cystatins varies from body fluid to body fluid, and in different compartments [Abrahamson M, et al: J. Biol. Chem. 1986; 261:11282]. In 1990, Abrahamsson et al. demonstrated that Cystatin C is produced at a stable rate and by all (or close to all) nucleated cells in the human body, being what is called a “householding” protein [Abrahamson M, et al: Biochem. J. 1990; 268:287-94].
Cystatin C—with a molecular mass of 13 kD—is freely filtered through the normal glomerular membrane of the kidney, but is then reabsorbed and catabolized in the “proximal tubuli”, the compartment of the kidneys that re-absorbs most of the peptides and smaller proteins that pass the glomerular membrane. In this way the kidneys preserve these proteinaceous materials for the body and hinder loss to the urine (Jacobsson B, et al: Histopathology 1995; 26:559-64.) [Heyms S B, et al: Am. J. Clin. Nutr. 1983; 37: 478].
Creatinine, until now the mostly used marker for glomerular filtration rate (GFR), is a small molecular weight molecule produced in muscles cell. The rate of formation and secretion of creatinine is therefore closely linked to the muscular mass of the body. It is well known that the muscular mass of an individual in a population varies considerably.
Elderly people often have a low muscular mass compared to the body mass, also many patients lose muscular mass as their diseases progress. Children generally have a different relative muscular mass compared to adults, and men have in average a higher relative muscular mass than women. Therefore, when serum creatinine concentration is used as a marker for glomerular filtration rate, the serum creatinine value can be found within the reference range even when a 50% reduction in glomerular filtration rate has taken place [Shemesh O, et al: Kidn. Int. 1985; 28: 830-8]. Also diet influences the serum level of creatinine significantly, especially a protein rich diet [Perrone R D, et al: Clin. Chem. 1992; 38: 1933-53].
Serum and plasma creatinine measurements are not reliable, “gold standard methods” for measurement of glomerular filtration rate. The use of intravenous injections of radio labeled substances like Cr-51-EDTA or Tc-99m-DTPA, or injections of iodinated agents like iohexol, have therefore gained some popularity. These methods are expensive, time-consuming and injections are necessary—and often repeated blood sampling. In the publication “Simple Cystatin C-Based Prediction Equations for Glomerular Filtration Rate Compared with the Modification of Diet in Renal Disease Prediction Equation for Adults and the Scwartz and the Counahan-Barratt Prediction Equations for Children”, by Grubb & al. in Clinical Chemistry 51:8, 1420-1431, it was demonstrated that simple Cystatin C measurements in blood can replace complicated methods of measuring the glomerular filtration rate.
Cystatin C measurement originally used classical biochemical methods, but soon moved over to turbidimetric and nephelometric immunoassays. The Dade-Behring Company pioneered nephelometric measurement methods. See also CoII, E & al. Am. J. Kidney Disease 2000; 36, 2934. The nephelometric methods are very reliable. The disadvantage of nephelometric methods is that the instruments themselves have rather slow processing speed compared to automated spectrophotometers based on light transmission. Nephelometers produced by Dade Behring, typically the BNII nephelometer and ProSpec nephelometers, and Beckman Instruments, have gained substantial market penetration, but are much less widespread than instruments based on transmission measurements. Nephelometers also have a much lower sample throughput than the large automated instruments based on transmission measurements.
H. Stone & al. in the article “Analytical performance of a particle-enhanced nephelometric immunoassay for serum Cystatin C using rate analysis”, in Clinical Chemistry Vol 8, pp 1482-85, 2001, presented a rate nephelometric method. However, nephelometric instruments have limited capacity for a large number of samples, and the number of nephelometric instruments is limited. Therefore there was a demand to move to absorption spectrophotometric-homogeneous instruments, since such instruments are more abundant and have a higher capacity.
Dako Cytomation, Denmark, has pioneered the field of turbidimetric Cystatin C immunoassays. The state of the art is described in the following articles: “Cystatin C—The marker of choice for renal function testing” by C. Schmidt in European Clinical Laboratory, 10 Feb. 2004. “New improved automated particle enhanced turbidimetric immunoassay for quantitative determination of human Cystatin C in serum and plasma” by C. Schmidt, C. Kjøller and K Grønkjær, a presentation downloaded from the Dako Cytomation AS' web site July 2005 (incorporated by reference).
Turbidimetric measurements are—however—disturbed by turbidity in samples, as described in the article “Turbidity in immunoturbidimetric assays” by Dahr & al, in Ann. Clin. Biochem. Vol 27, p. 509, 1990. At the 29th Nordic Congress of Clinical Chemistry, Malmo, Sweden, 24-27 Apr., 2004, A. Grubb presented results on interference from triglycerides in serum and plasma samples on the results from Cystatin C measurements. There is therefore still a need for improvements on the turbidimetric immunoassay for human Cystatin C.
An alternative to homogeneous immunoassay systems would be a separation-based non-homogeneous immunoassay systems. The advantage of non-homogeneous immunoassay systems is characterized by a separation step with washing during the performance of the assay. The advantage of these systems is that they—by means of the washing—remove any interfering substances. Furthermore, it allows washing away non-bound antibodies and non-bound enzymes or fluorescent or colored or other signal-giving moieties. These methods are generally non-competitive since the substance to be measured—hereinafter called the analyte—does not have to compete with labeled analogues for the binding to the antibodies. This aspect, and the use of washing steps, makes it possible to use immobilized antibodies and labeled antibodies in excess, rendering a high sensitivity and usually a high level of accuracy, especially if high quality antibodies are used.
There are numerous textbooks on solid phase and other non-homogeneous immunoassay systems. Big automated systems are commercially available. Abbott Diagnostic
Division, US, sells reagents for ImX, AxSYM and other automated non-homogeneous immunoassay instruments; Roche Diagnostics, Germany, sells reagents for Elecsys and other non-homogeneous immunoassay systems; and there are numerous other suppliers of non-homogeneous immunoassay systems and reagents. A still very popular non-homogeneous immunoassay system is the so-called microtitre immunoassay systems. These systems are slow but reliable and inexpensive. The general immunoassay literature gives a lot of information on these systems, which are well known to those skilled in the art.
The disadvantage of non-homogeneous immunoassay systems is the lack of capacity for a high number of assays per time unit, i.e. per hour running time. The methods involve separation and washing steps, which—although automated—occupy instrument-running time. Furthermore, since solid phases are involved, special devices or solid phases are often used, which have a certain production price, even when mass-produced. High through-put and speed of result generation have become more and more important in recent years, due to the ever increasing number of tests to run. There is a demand to move assays from non-homogeneous, often solid phase based, immunoassay technologies, to homogeneous immunoassay systems.
Homogeneous immunoassay systems are simple in construction and have a higher throughput capacity. The reagents are mixed, incubated and measured, either during or after incubation. Endpoint signals or differences in signal between different time points or continuous kinetic measurements are used. Signals may be measurements of fluorescence, color or opacity (turbidimetry). In particular, large automated instruments for measurements of color and/or opacity/turbidimetry have been very successful. The Hitachi Company in Japan pioneered this large-scale automation, working together with Roche, but also numerous other manufacturers including Olympus and Bayer supply such instruments and reagents. These instruments are automated multi-well spectrophotometers measuring transmission of light through a mixture of the sample and reagents at different wavelengths and at different time points. Since the turbidimetric Cystatin C immunoassays run on such homogeneous high capacity instruments, this is another reason why high quality turbidimetric Cystatin C assays are in demand.
Most homogeneous immunoassay systems are based on mixing the sample with reagent solutions comprising a well-controlled concentration of labeled analyte or analyte analogue, and antibodies having specific affinity for the analyte. The analyte molecules in the sample compete with the labeled analyte or analyte analogue, and a signal is generated. The vast immunoassay literature teaches the use of different labels and analogues. Here it should be noted that—so far—there has been limited success using homogeneous immunoassay methods for high molecular weight analytes (molecular mass above 4000) generating colored signals. (There has been success using fluorescent techniques, e.g. proximity assays, but there are not many high throughput automated fluorometers available, and therefore these technologies do not serve the purpose of the present invention). The EMIT technology from Syva and the CDEIA technology from Microgenics, both companies located in California, have been successful with color signals in homogeneous immunoassays for low molecular mass analytes. For high molecular mass analytes, particle enhanced turbidimetric measurements have been the most successful technology for the automated multi-well spectrophotometers measuring transmission of light through the mixture of the sample and the reagents. Turbidimetric methods are therefore preferred for higher molecular weight analytes. General descriptions of turbidimetric methods are found in numerous immunoassay textbooks.
Why are high precision measurements of Cystatin C necessary? From the perspective of biology, the literature on the biology of Cystatin C and creatinine, as well as the meta-analysis cited above, point in the direction of the use of Cystatin C instead of creatinine to monitor and diagnose reduced glomerular filtration rate. However, although the biological argument is in favor of using Cystatin C, the biological advantage is only of importance if the accuracy and precision of the Cystatin C analysis is good. The accuracy and precision of creatinine measurements is excellent, and the medical advantage of analyzing Cystatin C instead of creatinine is easily lost if the accuracy or precision of the Cystatin C immunoassay methods is too low.
Newman & al, Kidney Int. Vol 47 (1995), pp 312-318, teach an optimized assay using 77 nm (before coating) particles covalently coupled to rabbit anti-human Cystatin C immunoglobulin fraction. They do not teach the use of affinity purified antibodies, and they say nothing about the concentration of binding antibodies in the immunoglobulin fraction they use. They teach from 5 to 30% antibody weight per particle weight, optimized at 5% , however say nothing about the fraction of antibodies having affinity for Cystatin C in the immunoglobulin fraction. Typically, a good conventional antiserum comprises 5 to 15% active antibodies of the total antibodies in immunoglobulin fraction, which would imply 0.25 to 0.75 wt.-% of active antibodies per weight of particles. This is very low, that is probably the reason why Newman et al had to go all the way down to 340 nm to obtain a good signal. This is also why they observed a background signal being very high. Newman et al state in Table 1 that the particles are diluted down (how much is not stated) to give a starting absorbance of 0.7 at 0.74 cm light path length, which is still very high (more than an absorbance of 0.9 at 1 cm light path length, which is most often used), and therefore will lead to a very high background in the assay. This is maybe the main reason why the method of Newman et al never was commercialized or taken into clinical use, and it teaches away from using low wavelengths in Cystatin C particle enhanced turbidimetric immunoassay. All commercial products for turbidimetric measurements of Cystatin C on the market therefore use wavelengths above 500 nm.
A problem to be solved by the present invention is to provide an immuno-turbidimetric
Cystatin C immunoassay with improved accuracy and/or precision and/or less interference from lipids and hemoglobin in test sample if compared to corresponding commercially available turbidimetric assays.
Another drawback of the prior art of turbidimetric measurements of Cystatin C is the low rate of change in turbidimetry, which leads to long assay time and/or high imprecision. A further problem to be solved by the present invention is therefore, to provide a faster turbidimetric measurements of Cystatin C.